The Phosphoenolpyruvate:glycose phosphotransferase system (PTS) protein IIIGlc was investigated by nuclear magnetic resonance (NMR) techniques in order to better understand the function of the protein. Studies were carried out to characterize (1) the three-dimensional (3D) structure of phosphorylated IIIGlc and (2) the chemistry of the active site histidines. (1) 3D protein structure. Although the 3D protein structure of IIIGlc had been characterized in solution, by NMR, and in the crystalline state, by X-ray diffraction, the effect of phosphorylation on the structure of IIIGlc was unknown. Phosphorylation of IIIGlc affects binding of the protein to sugar permeases, and thereby regulates the uptake of specific sugars by the cell. A regeneration system was developed that maintained IIIGlc in the phosphorylated state for many days. This made it possible to perform multidimensional NMR experiments on P-IIIGlc which yielded information about its 3D structure. It was found that the structure of P-IIIGlc was essentially identical to that of IIIGlc. This result indicates that the placement of a negatively charged phosphate in the hydrophobic active site of the protein, rather than a conformation change, causes the change of the binding of IIIGlc to sugar permeases. (2) Histidine chemistry. IIIGlc contains two active site His residues. Using NMR we have shown that (a) both His residues are uncharged and have anomalously low pKa values, < 5, and (b) the two His residues are in the opposite tautomeric forms. The NMR results are the first information about the chemical state of the active site His residues and provide a basis for understanding the mechanism of phosphoryl transfer in the PTS. The significance of this project lies in its potential for providing a rational quantitative understanding of the function of the PTS. The PTS has essential and diverse physiological roles in many bacterial cells, including those responsible for dental caries.